Zinc is an essential metallic element that is present in the human body in the largest amount next to iron. Most zinc ions in cells are strongly coupled to proteins and are involved in the maintenance of structure or in the expression of function of the protein. Various reports have been also made on the physiological role of free zinc ions which are present in the cell in a very small quantity (generally at a level of μM or lower). In particular, zinc ions are considered to be significantly involved in one type of cell death, i.e., apoptosis, and it is reported that zinc ions accelerate senile plaque formation in Alzheimer's disease.
A compound (a fluorescent probe for zinc), which specifically traps a zinc ion to form a complex and emits fluorescence upon the formation of the complex, has been conventionally used to measure zinc ions in tissue. For example, TSQ (Reyes, J. G., et al., Biol. Res., 27, 49, 1994), Zinquin ethyl ester (Tsuda, M. et al., Neurosci., 17, 6678, 1997), Dansylaminoethylcyclen (Koike, T. et al., J. Am. Chem. Soc., 118, 12686, 1996), and Newport Green (a catalog of Molecular Probes, Inc.: “Handbook of Fluorescent Probes and Research Chemicals” 6th Edition by Richard P. Haugland pp. 531-540) have been used practically as fluorescent probes for zinc.
The measurement using TSQ, Zinquin, or Dansylaminoethylcyclen, however, requires the use of a short wavelength excitation light (an excitation wavelength of 367 nm, 368 nm, and 323 nm, respectively). Accordingly, when these fluorescent probes for zinc are used for measurement in living systems, the short wavelength excitation light may cause damages of cells (Saibou Kougaku (Cell Technology), 17, pp. 584-595, 1998). A problem also arises that the measurement may be readily influenced by autofluorescence generated from cell systems, per se (fluorescence emitted by NADH or flavins). Further, Dansylaminoethylcyclen suffers from a disadvantage: When the concentration is determined using the reagent, the fluorescence intensities are vary greatly depending on differences in environment such as solvents, whether a solubility in water or in lipid is extra-, intra-cell or cell membrane; or the like (Tanpakushitsu-Kakusan-Kouso (Protein, Nucleic Acid and Enzyme), extra number, 42, pp. 171-176, 1997). TSQ has a problem that even distribution in the whole cell is difficult due to its high lipophilicity. Newport Green has low affinity for zinc ions and fails to achieve practical measurement sensitivity, although the agent enables measurement with a long wavelength excitation light. Therefore, the development of a fluorescent probe for zinc has been desired that can measure zinc ions with high sensitivity without damaging cells.
The inventors of the present invention conducted various studies to provide a fluorescent probe for zinc with high sensitivity. As a result, they found that a compound having a cyclic amine or a polyamine as a substituent has high specificity with zinc ions, and by trapping zinc ions, the compound forms a complex which emits strong fluorescence with a excitation light in longer wavelength range (Japanese Patent Application No. (Hei) 11-40325). In addition, the inventors further conducted studies and succeeded in providing a fluoresceine derivative by which zinc in the living organism can be measured with very high accuracy and sensitivity (International Publication WO 01/62755). However, this fluoresceine derivative has extremely high affinity for a zinc ion, and this causes a difficulty in accurately measuring zinc ions in a high concentration, although the derivative was excellent in detection of zinc ions in a low concentration. Therefore, it has been desired to provide a means which can accurately measure the concentration of zinc ions in a broader range.